Methods for prevention and treatment of inflammation using anti-chemokine antibodies

ABSTRACT

It is possible to inhibit inflammatory processes by administration of antibodies to chemokines. Identification of chemokines which are over-produced makes it possible to block specific chemokine activity using antibodies to the over-expressed chemokines.

This application is a continuation application of U.S. patentapplication Ser. No. 10/712,393, filed on Nov. 14, 2003, which claimspriority of U.S. Provisional Application No. 60/426,350, filed on Nov.15, 2002.

This work was partially supported by the U.S. Government. Hence, certainrights belong to the government.

FIELD

This invention relates to antibodies or the use of antibodies directedagainst certain chemokines. The antibodies block high-affinityinteractions leading to inflammation.

BACKGROUND

Despite recent advances in studies related to the inflammation process,therapies for treatment of chronic inflammatory diseases have remainedelusive. This is perhaps a result of the many and complex factors in thehost that initiate and maintain inflammatory conditions. Currenttherapies have disadvantages associated with them, including thesuppression of the immune system that can render the host moresusceptible to bacterial, viral and parasitic infections. For example,use of steroids is a traditional approach to chronic inflammationtreatment. Such treatment can lead to changes in weight and suppressionof protective immunity. Advances in biotechnology have promoted thedevelopment of targeted biologicals with fewer side effects. To improveinflammatory disease treatment, technologies that alter and control thefactors generated by cells of both innate and adaptive immunity systemsneed to be developed.

Host cells have surface receptors that associate with ligands to signaland regulate host cell activities. Administration of anti-TNF-α antibodyor soluble TNF-α receptor has been shown to inhibit inflammatorydiseases. Unfortunately, the side effects associated with this treatmentcan result in an increased risk of infections (e.g., tuberculosis) andother adverse reactions by mechanisms not fully understood. Similarly,antibody therapies focused on membrane bound molecules like CD40 have apropensity for inhibiting inflammation and graft-host diseases. Whileother targeted host cell therapies to prevent inflammatory diseases arebeing developed, there is no known single surface or secreted factorthat will stop all inflammatory diseases. Consequently, the developmentof therapies to exploit newly identified specific host cell targets isrequired.

A variety of pathogens or toxins activate macrophages, neutrophils, Tcells, B cells, monocytes, NK cells, Paneth and crypt cells, as well asepithelial cells shortly after entry into the mucosa. Chemokines are asuperfamily of small, cytokine-like proteins that are resistant tohydrolysis, promote neovascularization or endothelial cell growthinhibition, induce cytoskeletal rearrangement, activate or inactivatelymphocytes, and mediate chemotaxis through interactions withG-protein-coupled receptors. Chemokines can mediate the growth andmigration of host cells that express their receptors. The cellularmechanisms responsible for the function of chemokines are often, but notentirely, Ca²⁺ flux dependent and pertussis toxin-sensitive. However,the precise mechanisms for chemokine-mediated events are not known.

SUMMARY

The present invention provides a means of inhibiting inflammation byadministering anti-chemokine antibodies. Exemplified are anti-CXCL9,-CXCL10, -CXCL11, -CCRL1, -CCRL2, -CCR5, -CCL1, -CCL2, -CCL3, -CCL4,-CCL4L1, -CCL5, -CCL7, -CCL8, -CCL14-1, -CCL14-2, -CCL14-3, -CCL15-1,-CCL15-2, -CCL16, -CCL19, -CCL23-1, -CCL23-2, -CCL24, -CCL26, -CCR6,-CCL20, and -CCL25, -CCL25-1, -CCL25-2 antibodies. These chemokines areknown. The Genbank assession numbers are provided herein. The inventionutilizes antibodies or functional fragments thereof that bind to thetarget chemokines. The antibodies or antigen-binding fragment(s) bind toepitope(s) or peptide(s) that consists of 10 to 15 amino acids fromsequences 1 through 30 (accession numbers in NIH-NCBI Genebank givenbelow) of the target chemokines. Antibodies which act as modulators canadministered mucosally or systemically.

The antibody, antibodies, or antigen-binding fragment or fragments canbe isolated from the serum of immunized mammalian hosts, from culturesof immortalized cell lines and tissues such as hybridomas,lymphoblastoid or cells generated by methods of recombinant moleculebiology. For increased effectiveness, fragments may be conjugated orlinked to other peptides, proteins, nucleic acid sequences, vitamins,complex or simple carbohydrates, or other suitable carrier molecules.

Antibodies or antigen-binding fragments with specificity for functionalmutant or variant mammalian chemokines are appropriate. These mutationsor polymorphisms occur in nature or can be induced by recombinantmolecular biological methods to generate single, multiple, or continuousamino acid residues, described in sequences 1 through 30, that aredeleted, added, and/or substituted for other or no amino acids.

The mucosal means of application include oral, intranasal, ocular,intravaginal, rectal, and/or intraurethral administration in liquid orparticulate form or on solid supports. Systemic means of applicationinclude parenteral, intravenous, or intramuscular administration inliquid or particulate form.

DETAILED DESCRIPTION

The invention provides methods of identifying, evaluating and treating asubject suffering from conditions arising from inflammatory processes,including anaphylaxis and septic shock, arthritis (e.g., osteoarthritis,rheumatoid, psoriatic), asthma, allergies (e.g., drug, insect, plant,food), atherosclerosis, delayed type hypersensitivity, dermatitis,diabetes (e.g., mellitus, juvenile onset), graft rejection, inflammatorybowel diseases (e.g., Crohn's disease, ulcerative colitis, enteritis),interstitial cystitis, multiple sclerosis, myasthemia gravis, Grave'sdisease, Hashimoto's thyroiditis, pneumonitis, prostatitis, psoriasis,nephritis, pneumonitis, chronic obstructive pulmonary disease, chronicbronchitis rhinitis, spondyloarthropathies, scheroderma, systemic lupuserythematosus, or throiditis using the said embodiments. As a result oneor more of inflammatory processes including host cell migration orexcretion.

It has now been demonstrated, using as examples anti-CXCL9, -CXCL10,-CXCL11, -CCRL1, -CCRL2, -CCR5, -CCL1, -CCL2, -CCL3, -CCL4, -CCL4L1,-CCL5, -CCL7, -CCL8, -CCL14-1, -CCL14-2, -CCL14-3, -CCL15-1, -CCL15-2,-CCL16, -CCL19, -CCL23-1, -CCL23-2, -CCL24, -CCL26, -CCR6, -CCL20, and-CCL25, -CCL25-1, -CCL25-2 antibodies, that it is possible to inhibitthe inflammatory cell activation, migration or chemotaxis of the byinflammatory processes.

Materials and Methods

Primer Design

Messenger RNA sequences for CXCL9, CXCL10, CXCL11, CCRL1, CCRL2, CCR5,CCL1, CCL2, CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL14-1, CCL14-2,CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, CCL23-1, CCL23-2, CCL24, CCL26,CCR6, CCL20, and CCL25, CCL25-1, CCL25-2 were obtained from the NIH-NCBIgene bank database (Sequences 31 through 60, accession numbers givenlater). Primers were designed using the BeaconJ 2.0 computer program.Thermodynamic analysis of the primers was conducted using computerprograms: Primer PremierJ and MIT Primer 3. The resulting primer setswere compared against the entire human genome to confirm specificity.

Real Time PCR Analysis

Lymphocytes or inflamed tissues were cultured in RMPI-1640 containing10% fetal calf serum, 2% human serum, supplemented with non-essentialamino acids, L-glutamate, and sodium pyruvate (complete media).Additionally, primary inflammatory and normal-paired matched tissueswere obtained from clinical isolates (Clinomics Biosciences, Frederick,Md. and UAB Tissue Procurement, Birmingham, Ala.). Messenger RNA (mRNA)was isolated from 10⁶ cells using TriReagent (Molecular Research Center,Cincinnati, Ohio) according to manufacturers protocols. Potentialgenomic DNA contamination was removed from these samples by treatmentwith 10 U/μl of RNase free DNase (Invitrogen, San Diego, Calif.) for 15minutes at 37° C. RNA was then precipitated and resuspended in RNASecure (Ambion, Austin, Tex.). cDNA was generated by reversetranscribing approximately 2 μg of total RNA using Taqman7 reversetranscription reagents (AppliedBiosystems, Foster City, Calif.)according to manufacturers protocols. Subsequently, cDNA's wereamplified with specific human cDNA primers, to CXCL9, CXCL10, CXCL11,CCRL1, CCRL2, CCR5, CCL1, CCL2, CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8,CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, CCL23-1,CCL23-2, CCL24, CCL26, CCR6, CCL20, and CCL25, CCL25-1, CCL25-2, usingSYBR7 Green PCR master mix reagents (Applied Biosystems) according tomanufacturers protocol. The level of copies of mRNA of these targetswere evaluated by real-time PCR analysis using the BioRad Icycler andsoftware (Hercules, Calif.).

Anti-Sera Preparation

The 15 amino acid peptides from chemokines CXCL9, CXCL10, CXCL11, CCRL1,CCRL2, CCR5, CCL1, CCL2, CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL14-1,CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, CCL23-1, CCL23-2,CCL24, CCL26, CCR6, CCL20, and CCL25, CCL25-1, CCL25-2 (Sequences 1through 30) were synthesized (Sigma Genosys, The Woodlands, Tex.) andconjugated to hen egg lysozyme (Pierce, Rockford, Ill.) to generate theantigens for subsequent immunizations for anti-sera preparation ormonoclonal antibody generation. The endotoxin levels of chemokinepeptide conjugates were quantified by the chromogenic Limulus amebocytelysate assay (Cape Cod, Inc., Falmouth, Miss.) and shown to be <5 EU/mg.100 μg of the antigen was used as the immunogen together with completeFreund's adjuvant Ribi Adjuvant system (RAS) for the first immunizationin a final volume of 1.0 ml. This mixture was administered in 100 mlaliquots on two sites of the back of the rabbit subcutaneously and 400ml intramuscularly in each hind leg muscle. Three to four weeks later,rabbits received 100 μg of the antigen in addition to incompleteFreund's adjuvant for 3 subsequent immunizations. Anti-sera werecollected when antibody titers reached 1:1,000,000. Subsequently, normalor anti-sera were heat-inactivated and diluted 1:50 in PBS.

Monoclonal Antibody Preparation

The 15 amino acid peptides from chemokines CXCL9, CXCL10, CXCL11, CCRL1,CCRL2, CCR5, CCL1, CCL2, CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL14-1,CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, CCL23-1, CCL23-2,CCL24, CCL26, CCR6, CCL20, and CCL25, CCL25-1, CCL25-2 (Sequences 1through 30) were synthesized (Sigma Genosys) and conjugated to hen egglysozyme (Pierce) to generate the antigens for subsequent immunizationsfor anti-sera preparation or monoclonal antibody generation. Theendotoxin levels of chemokine peptide conjugates were quantified by thechromogenic Limulus amebocyte lysate assay (Cape Cod, Inc., Falmouth,Miss.) and shown to be <5 EU/mg. 100 μs of the antigen was used as theimmunogen together with complete Freund's adjuvant Ribi Adjuvant system(RAS) for the first immunization in a final volume of 200 μl. Thismixture was subcutaneously administered in 100 μl aliquots at two sitesof the back of a rat, mouse, or immunoglobulin-humanized mouse. Twoweeks later, animals received 100 μg of the antigen in addition toincomplete Freund's adjuvant for 3 subsequent immunizations. Serum werecollected and when anti-CXCL9, -CXCL10, -CXCL11, -CCRL1, -CCRL2, -CCR5,-CCL1, -CCL2, -CCL3, -CCL4, -CCL4L1, -CCL5, -CCL7, -CCL8, -CCL14-1,-CCL14-2, -CCL14-3, -CCL15-1, -CCL15-2, -CCL16, -CCL19, -CCL23-1,-CCL23-2, -CCL24, -CCL26, -CCR6, -CCL20, and -CCL25, -CCL25-1, -CCL25-2antibody titers reached 1:2,000,000, hosts were sacrificed andsplenocytes were isolated for hybridoma generation.

B cells from the spleen or lymph nodes of immunized hosts were fusedwith immortal myeloma cell lines (e.g., YB2/0). Hybridomas were nextisolated after selective culturing conditions (i.e., HAT-supplementedmedia) and limiting dilution methods of hybridoma cloning. Cells thatproduce antibodies with the desired specificity were selected usingELISA. Hybridomas from normal rats or mice were humanized with molecularbiological techniques in common use. After cloning a high affinity andprolific hybridoma, antibodies were isolated from ascites or culturesupernatants and adjusted to a titer of 1:2,000,000 and diluted 1:50 inPBS.

Anti-Sera or Monoclonal Antibody Treatment

Knockout or transgenic mice (8 to 12 weeks old, Charles RiverLaboratory, Wilmington, Mass.) that spontaneous—or when treated—developinflammatory diseases were treated with 200 μl intraperitonealinjections of either anti-sera or monoclonal antibodies specific foreach of the chemokines every three days. The inflammatory disease stateof the host was next monitored for progression or regression of disease.

Cytokine Analysis by ELISA

The serum level of IL-2, -IL-6, -TNF-α, and -IFN-γ were determined byELISA, following the manufacturers instructions (E-Biosciences, SanDiego, Calif.). Plates were coated with 100 μl of the respective captureantibody in 0.1 M bicarbonate buffer (pH 9.5) and incubated O/N at 4° C.After aspiration and washing with wash buffer, the wells were blockedwith assay diluent for 1 hour at RT. Samples and standards were addedand incubated for 2 hours at RT. Next, 100 μl of detection antibodysolutions were added and incubated for 1 hour. 100 μl of avidin-HRPsolution was added and incubated for 30 minutes. Subsequently, 100 μlTetramethylbenzidine (TMB) substrate solution was added and allowed toreact for 20 minutes. 50 μl of the stop solution was added and plateswere read at 450 nm. The cytokine ELISA assays were capable ofdetecting >15 pg/ml for each assay.

Cytokine Analysis by Multiplex Cytokine ELISA

The T helper cell derived cytokines, IL-1α, IL-1β, IL-2, IL-12, IFN-γ,TNF-α, in serum were also determined by Beadlyte mouse multi-cytokinedetection system kit provided by BioRad, following manufacturerinstructions. Filter bottom plates were rinsed with 100 μl of bio-plexassay buffer and removal using a Millipore Multiscreen Separation VacuumManifold System (Bedford, Mass.), set at 5 in Hg. IL-1α, IL-1β, IL-2;IL-12, IFN-γ, TNF-α beads in assay buffer were added into wells. Next,50 μl of serum or standard solution were added and the plates wereincubated for 30 minutes at RT with continuous shaking (setting 3) usinga Lab-Line Instrument Titer Plate Shaker (Melrose, Ill.), after sealingthe plates. The filter bottom plates were washed 2 times, as before, andcentrifuged at 300×g for 30 seconds. Subsequently, 50 μl of anti-mouseIL-1α, IL-1β, IL-2, IL-12, IFN-γ, TNF-α antibody-biotin reportersolution was added in each well followed by incubation with continuousshaking for 30 minutes followed by centrifugation at 300×g for 30seconds. The plates were washed 3 times with 100 μl of bio-plex assaybuffer as before. Next, 50 μl streptavidin-phycoerythrin solution wasadded to each well and incubated with continuous shaking for 10 minuteat RT. 125 μl of bio-plex assay buffer was added and Beadlyte readingswere measured using a Luminexl instrument (Austin, Tex.). The resultingdata was collected and calculated using Bio-plexl software (Bio-Rad).The cytokine Beadlyte assays were capable of detecting >5 pg/ml for eachanalyte.

Serum Amyloid Protein A (BAA) ELISA

The SAA levels were determined by ELISA using a kit supplied byBiosource International, (Camarillo, Calif.). Briefly, 50 μl ofSAA-specific monoclonal antibody solution was used to coat micro-titerstrips to capture SAA. Serum samples and standards were added to wellsand incubated for 2 hours at RT. After washing in the assay buffer, theHRP-conjugated anti-SAA monoclonal antibody solution was added andincubated for 1 hour at 37° C. After washing, 100 μlTetramethylbenzidine (TMB) substrate solution was added and the reactionwas stopped after incubation for 15 minutes at RT. After the stopsolution was added, the plates were read at 450 nm.

Histology and Pathology Scoring

Fixed tissues were sectioned at 6 μm, and stained with hematoxylin andeosin for light microscopic examination. The intestinal lesions weremulti-focal and of variable severity, the grades given to any section ofintestine took into account the number of lesions as well as theirseverity. A score (0 to 4) was given, based on the following criteria:(Grade 0) no change from normal tissue. (Grade 1) 1 or a few multi-focalmononuclear cell infiltrates, minimal hyperplasia and no depletion ofmucus. (Grade 2) lesions tended to involve more of the mucosa andlesions had several multi-focal, yet mild, inflammatory cell infiltratesin the lamina propria composed of mononuclear cells, mild hyperplasia,epithelial erosions were occasionally present, and no inflammation wasnoticed in the sub-mucosa. (Grade 3) lesions involved a large area ofmucosa or were more frequent than Grade 2, where inflammation wasmoderate and often involved in the sub-mucosa as well as moderateepithelial hyperplasia, with a mixture of mononuclear cells andneutrophils. (Grade 4) lesions usually involved most of the section andwere more severe than Grade 3 lesions. Additionally, Grade 4inflammations were more severe and included mononuclear cell andneutrophils; epithelial hyperplasia was marked with crowding ofepithelial cells in elongated glands. The summation of these scoreprovide a total inflammatory disease score per mouse. The disease scorecould range from 0 (no change in any segment) to a maximum of 12 withGrade 4 lesions of segments.

Data Analysis

SigmaStat 2000 (Chicago, Ill.) software was used to analyze and confirmthe statistical significance of data. The data were subsequentlyanalyzed by the Student's t-test, using a two-factor, unpaired test. Inthis analysis, treated samples were compared to untreated controls. Thesignificance level was set at p<0.05.

Results

Semiquantitative RT-PCR Identification of Molecular Targets

RT-PCR products obtained using CXCL9-, CXCL10-, CXCL11-, CCRL1-, CCRL2-,CCR5-, CCL1-, CCL2-, CCL3-, CCL4-, CCL4L1-, CCL5-, CCL7-, CCL8-,CCL14-1-, CCL14-2-, CCL14-3-, CCL15-1-, CCL15-2-, CCL16-, CCL19-,CCL23-1-, CCL23-2-, CCL24-, CCL26-, CCR6-, CCL20-, and CCL25-, CCL25-1-,CCL25-2-specific primer sets did not cross react with other gene targetsdue to exclusion of primers that annealed to host sequences 31-60. Theprimers used produced different size amplicon products relative thepolymorphisms that resulted in CCL4 versus CCL4L1, CCL14-1, CCL14-2,versus CCL14-3, CCL15-1 versus CCL15-2, CCL23-1 versus CCL23-2, andCCL25, CCL25-1, versus CCL25-2. To this end, RT-PCR analysis of tissuefrom subjects exhibiting anaphylaxis, arthritis (e.g., rheumatoid,psoriatic), asthma, allergies (e.g., drug, insect, plant, food),atherosclerosis, delayed type hypersensitivity, dermatitis, diabetes(e.g., mellitus, juvenile onset), graft rejection, inflammatory boweldiseases (e.g., Crohn's disease, ulcerative colitis, enteritis),multiple sclerosis, myasthemia gravis, pneumonitis, psoriasis,nephritis, rhinitis, spondyloarthropathies, scheroderma, systemic lupus,or throiditis revealed that CXCL9, CXCL10, CXCL11, CCRL1, CCRL2, CCR5,CCL1, CCL2, CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL14-1, CCL14-2,CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, CCL23-1, CCL23-2, CCL24, CCL26,CCR6, CCL20, and CCL25, CCL25-1, CCL25-2 were differentially expressedby inflammatory host cells.

In Vivo Inflammatory Disease Inhibition

Mammals that develop anaphylaxis, septic shock, arthritis (e.g.,rheumatoid, psoriatic), asthma, allergies (e.g., drug, insect, plant,food), atherosclerosis, bronchitis, chronic pulmonary obstructivedisease, delayed type hypersensitivity, dermatitis, diabetes (e.g.,mellitus, juvenile onset), graft rejection, Grave's disease, Hashimoto'sthyroiditis, inflammatory bowel diseases (e.g., Crohn's disease,ulcerative colitis, enteritis), interstitial cystitis, multiplesclerosis, myasthemia gravis, pneumonitis, psoriasis, nephritis,rhinitis, spondyloarthropathies, scheroderma, systemic lupuserythematosus, or throiditis were allowed to develop the inflammatorydisease of interest. Antibodies directed against CXCL9, CXCL10, CXCL11,CCRL1, CCRL2, CCR5, CCL1, CCL2, CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8,CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, CCL23-1,CCL23-2, CCL24, CCL26, CCR6, CCL20, or CCL25, CCL25-1, CCL25-2differentially affected the progression and regression of inflammatorydisease as determined by histological scoring and comparing pre- andpost-treatment serum levels of IFN-γ, IL-1α, IL-1β, IL-6, IL-12, TNF-α,amyloid protein A. Antibodies directed towards CXCL9, CXCL-10, CXCL11,CCRL1, CCRL2, CCR5, CCL1, CCL2, CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8,CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, CCL23-1,CCL23-2, CCL24, CCL26, CCR6, CCL20, or CCL25, CCL25-1, CCL25-2effectively lead to the both regression and impeding progression ofinflammatory disease as determined by histological scoring and comparingpre- and post-treatment serum levels of IFN-γ, IL-1α, IL-1β, IL-6,IL-12, TNF-α, amyloid protein A.

As indicated previously, the chemokines used in the methods of theinvention are known. The assession numbers for the protein sequences areas follows:

CXCL9, (ACCESSION# NM_(—)002416), CXCL10 (ACCESSION# NM_(—)001565),

CXCL11 (ACCESSION# NM_(—)005409), CCRL1 (ACCESSION# NM_(—)016557),

CCRL2 (ACCESSION# NM_(—)003965), CCR5 (ACCESSION# NM_(—)060579),

CCL1 (ACCESSION# NM_(—)002981), CCL2 (ACCESSION# NM_(—)002982),

CCL3 (ACCESSION# XM_(—)008450, NM_(—)002983),

CCL4 (ACCESSION# NM_(—)002984), CCL4L1 (ACCESSION# AY079147),

CCL5 (ACCESSION# NM_(—)002985), CCL7 (ACCESSION# NM_(—)006273),

CCL8 (ACCESSION# NM_(—)005623), CCL14-1 (ACCESSION# NM_(—)004166),

CCL14-2 (ACCESSION# NM_(—)032962),

CCL14-3 (ACCESSION# NM_(—)032963),

CCL15-1 (ACCESSION# NM_(—)032964),

CCL15-2 (ACCESSION# NM_(—)004167),

CCL16 (ACCESSION# NM_(—)004590), CCL19 (ACCESSION# NM_(—)006274),

CCL23-1 (ACCESSION# NM_(—)005064),

CCL23-2 (ACCESSION# NM_(—)145898),

CCL24 (ACCESSION# NM_(—)002991), CCL26 (ACCESSION# NM_(—)006072),

CCR6 (ACCESSION# U45984), CCL20 (ACCESSION# NM_(—)004591)

CCL25 (ACCESSION# 015444), CCL25-1 (ACCESSION# NM_(—)005624),

and CCL25-2 (ACCESSION# NM_(—)148888).

For the cDNA sequences, the following GenBank accession numbers apply:

CXCL9 (ACCESSION# NM_(—)002416), CXCL10 (ACCESSION# NM_(—)001565),

CXCL11 (ACCESSION# NM_(—)005409), CCRL1 (ACCESSION# NM_(—)016557),

CCRL2 (ACCESSION# NM_(—)003965), CCR5 (ACCESSION# NM_(—)000579),

CCL1 (ACCESSION# NM_(—)002981), CCL2 (ACCESSION# NM_(—)002982),

CCL3 (ACCESSION# XM_(—)008450, NM_(—)002983),

CCL4 (ACCESSION# NM_(—)002984), CCL4L1 (ACCESSION# AY079147),

CCL5 (ACCESSION# NM_(—)002985), CCL7 (ACCESSION# NM_(—)006273),

CCL8 (ACCESSION# NM_(—)005623), CCL14-1 (ACCESSION# 004166),

CCL14-2 (ACCESSION# NM_(—)032962), CCL14-3 (ACCESSION# NM_(—)032963),

CCL15-1 (ACCESSION# NM_(—)032964),

CCL15-2 (ACCESSION# NM_(—)004167),

CCL16 (ACCESSION# NM_(—)004590), CCL19 (ACCESSION# NM_(—)006274),

CCL23-1 (ACCESSION# NM_(—)005064),

CCL23-2 (ACCESSION# NM_(—)145898),

CCL24 (ACCESSION# NM_(—)002991), CCL26 (ACCESSION# NM_(—)006072),

CCR6 (ACCESSION# U45984), CCL20 (ACCESSION# NM_(—)004591),

CCL25 (ACCESSION# 015444), CCL25-1 (ACCESSION# NM_(—)005624),

As shown in the table, the particular chemokines which give rise toinflammatory diseases differ with the disease. They also differ amongindividuals. Hence, it is wise, when treating an individual, to identifythe particular chemokines which are increased in the tissues of thepatient. Using the antibodies produced against each of the chemokinesand exposing the tissue samples from the patient to the particularantibodies, then evaluating the amount of antibody/chemokine binding, itis possible to evaluate the level of expression for each chemokine andto administer to the patient the particular antibodies that will bindthe excessive chemokine. This tailored approach to treatment ofinflammatory disease is novel, and a particularly valuable aspect of theinvention.

TABLE 1 Chemokine, Chemokine Receptor and Inflammatory DiseaseAssociation (dependent of stage of disease) Disease Chemokine ChemokineReceptor Allergies CCL1, CCL2, CCL5, CCL7, CCL8, CCR3, CCR4, CCR8, CCR9(Skin, Food & Respiratory) CCL11, CCL13, CCL17, CCL22, CCL24, CCL25,CCL26 Asthma CCL3, CCL4, CCL5, CCL7, CCL8, CCR3, CCR4, CCR5, CCL11,CCL15, CCL17, CCL22, CCL24, CCL26, Septic Shock, Anaphylaxis CXCL1,CXCL2, CXCL3, CXCL5, CXCR1, CXCR2, CXCR3 CXCL6, CXCL7, CXCL8, CXCL9,CXCL10, CXCL11, CCL5 Arthritis CXCL9, CXCL10, CXCL11, CXCR3, CXCR4,CXCR5 (septic, rheumatoid, psoriatic) CXCL12, CXCL13 CCL20 CCR6 XCL1XCR1 CX3CL1 CX3CR1 Osteoarthritis CXCL1, CXCL2, CXCL3, CXCL5, CXCR1,CXCR2, CXCL6, CXCL7, CXCL8, CXCL12, CCR2, CCR5 CXCL13, CCL2, CCL3, CCL4,CCL7, CCL8, CCL13, CCL5, CCL18 Atherosclerosis CXCL1, CXCL2, CXCL3,CXCL4, CXCR1, CXCR2 CXCL5, CXCL8 CCL2, CCL3, CCL4, CCL8, CCR2, CCR8CCL12, CCL13, CCL17, CCL22 CX3CL1 CX3CR1 Dermatitis & Delayed-TypedCXCL9, CXCL10, CXCL11, CXCR3 Hypersensitivity CCL2, CCL3, CCL4, CCL5,CCR4, CCR5, CCR6, CCR10 CCL17, CCL20, CCL22, CCL27 Diabetes CXCL9,CXCL10, CXCL11, CXCR3 CCL2, CCL9 CCR2, CCR4 CX3CL1 CX3CR1 Graftrejection CXCL9, CXCL10, CXCL11, CXCR3 CCL3, CCL4, CCL5 CCR5 XCL1 XCR1Inflammatory Bowel Diseases CXCL9, CXCL10, CXCL11, CXCR3 CCL3, CCL4,CCL5 CCR5 Interstitial Cystitis CXCL9, CXCL10, CXCL11, CXCR3 CCL3, CCL4,CCL5 CCR5 Multiple Sclerosis CXCL9, CXCL10, CXCL11, CXCR3 CCL3, CCL4,CCL5, CCL7, CCR1, CCR5 CCL14, CCL15, CCL23 Myasthemia gravis, Grave'sCXCL9, CXCL10, CXCL11, CXCR3 disease, & Hashimoto thyroiditis CCL3,CCL4, CCL5 CCR5 XCL1 XCR1 Nephritis & Systemic Lupus CXCL9, CXCL10,CXCL11, CXCR3, CXCR5 Erthematosus CXCL13 CCL2, CCL3, CCL4, CCL5, CCL8,CCR2, CCR4 CCL12, CCL13, CX3CR1 CX3CL1 Pneumonitis, Chronic ObstructiveCXCL1, CXCL2, CXCL3, CXCL5, CXCR2, CXCR3 Pulmonary Disease, & ChronicCXCL7, CXCL8 Bronchitis CCL3, CCL5, CCL7, CCL8, CCR3 CCL11, CCL13,CCL24, CCL26.

The method consists of 1) exposing tissues from a mammal suffering fromand inflammatory condition to several differing antibodies which bind tospecific chemokines, 2) identifying level of expression of eachchemokine based on binding of the each of the differing antibodies, and3) administering to said mammal those specific antibodies which bind tothe over-expressed chemokines. Additionally, the level of expression ofany particular chemokine may be determined using PCR, as disclosedherein.

Antibodies of the invention may be administered directly to targettissue. For example, compositions containing the compositions containingantibodies as prepared under the heading “anti-sera preparation” can beadministered intravenously, rectally vaginally, intrathecally, byinhalation, transvaginally, transurethrally or directly to tissue duringsurgery. The anti-sera preparations may also be placed on a solidsupport such as a sponge or gauze for administration to the mucosa. Theantibodies may also be administered using microspheres or liposomes. Thecompositions for administration are prepared in the usualpharmaceutically acceptable carriers such as saline, water, bufferedsaline, glucose in saline, etc.

Liquid compositions will most often be prepared containing 0.1 to 1000μg of antibody in each ml of solution. All compositions will beadministered in an inflammatory inhibiting effective amount with theamount of dosage given depending on the size and condition of theindividual in need of anti-inflammatory treatment.

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the components and steps in any sequence which iseffective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

1-15. (canceled)
 16. A method of treating a mammal with inflammatorydisease, comprising: a) exposing a tissue sample from said mammal to anantibody which binds specifically to CXCL9, or an antibody which bindsspecifically to CXCL11, or both antibodies; b) determining a level ofexpression of CXCL9, or CXCL11 or both based on binding of said antibodyto said tissue sample; and c) if over-expression of CXCL9 or CXCL11 orboth are detected in said tissue sample of said mammal, administering tosaid mammal an effective amount of an antibody which binds specificallyto CXCL9, or an antibody which binds specifically to CXCL11, or bothantibodies for the treatment of said inflammatory disease.
 17. Themethod of claim 16, wherein said mammal is a human.
 18. The method ofclaim 16, wherein in step c, said antibody or antibodies are humanizedantibodies.
 19. The method of claim 16, wherein in step c, said antibodyor antibodies are chimeric antibodies.
 20. The method of claim 16,wherein in step c, said antibody or antibodies are administered directlyinto an inflamed tissue.
 21. The method of claim 16, wherein in step c,said antibody or antibodies are administered parenterally.
 22. Themethod of claim 16, wherein in step c, said antibody or antibodies areadministered systemically.
 22. The method of claim 16, wherein in stepc, said antibody or antibodies are administered in a liquid carrier. 23.The method of claim 16, wherein in step c, said antibody or antibodiesare administered on a solid support.
 24. The method of claim 16, whereinsaid inflammatory disease is selected from the group consisting ofanaphylaxis, septic shock, arthritis, asthma, allergies,atherosclerosis, bronchitis, chronic pulmonary obstructive disease,delayed type hypersensitivity, dermatitis, diabetes, graft rejection,Grave's disease, Hashimoto's thyroiditis, inflammatory bowel diseases,interstitial cystitis, multiple sclerosis, myasthemia gravis,pneumonitis, psoriasis, nephritis, rhinitis, spondyloarthropathies,scheroderma, systemic lupus erythematosus, and thyroiditis.
 25. A methodof treating a mammal with inflammatory disease, comprising: a)determining the level of CXCL9 or CXCL11 or both in a tissue sample insaid mammal using PCR; and b) if over-expression of CXCL9 or CXCL11 orboth is detected in said tissue sample of said mammal, administering tosaid mammal an effective amount of an antibody which binds specificallyto CXCL9, an antibody which binds specifically to CXCL11, or bothantibodies for the treatment of said inflammatory disease.
 26. Themethod of claim 25, wherein said antibody or antibodies are administereddirectly into an inflamed tissue.
 27. The method of claim 25, whereinsaid antibody or antibodies are administered systemically.
 28. Themethod of claim 25, wherein in step c, said antibody or antibodies areadministered parenterally.
 29. The method of claim 25, wherein saidantibody or antibodies are administered on a solid support.
 30. Themethod of claim 25, wherein said antibody or antibodies are human orhumanized antibodies.
 31. The method of claim 25, wherein in step c,said antibody or antibodies are chimeric antibodies.
 32. The method ofclaim 25, wherein said inflammatory disease is selected from the groupconsisting of anaphylaxis, septic shock, arthritis, asthma, allergies,atherosclerosis, bronchitis, chronic pulmonary obstructive disease,delayed type hypersensitivity, dermatitis, diabetes, graft rejection,Grave's disease, Hashimoto's thyroiditis, inflammatory bowel diseases,interstitial cystitis, multiple sclerosis, myasthemia gravis,pneumonitis, psoriasis, nephritis, rhinitis, spondyloarthropathies,scheroderma, systemic lupus erythematosus, and thyroiditis.